Benzyl methacrylate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Metabolic stability of the test item was analysed using pooled liver S9 fractions from male Sprague Dawley (SD) rats. Based on the knowledge gained during method development, in the present metabolic stability study of BNMA the following test conditions were used: 20 μM of BNMA were incubated in glass vials with 0.5 mg/ml S9 fractions and after 0, 2, 5,10,15 and 30 minutes samples were collected for analytical detection via LC-MS.

Two types of negative control (NC; n=3) i.e. heat-inactivated S9 fraction and pure assay buffer w/o S9 mix, respectively, were run in parallel to the experimental incubations to verify that any apparent loss of test article in the assay incubation was due to metabolism.

As positive control 1 μM verapamil was incubated in parallel to the test item (n=3), and the depletion of the compound was monitored to demonstrate the enzymatic activity of the S9 fractions. Positive control samples were taken after 0 and 30 minutes.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl methacrylate

EC Number:

219-674-4

EC Name:

Benzyl methacrylate

Cas Number:

2495-37-6

Molecular formula:

C11H12O2

IUPAC Name:

benzyl methacrylate

Test material form:

liquid

Specific details on test material used for the study:

Name of test material: Benzyl methacrylate

Radiolabelling:

no

Test animals

Species:

other: Rat liver S9 fractions

Strain:

Sprague-Dawley

Sex:

male

Administration / exposure

Vehicle:

DMSO

Duration and frequency of treatment / exposure:

sampling after 0, 2, 5,10,15 and 30 minutes

Doses / concentrations

Dose / conc.:

20 other: µM

Remarks:

The final test concentration was determined during experimental development.

No. of animals per sex per dose / concentration:

not applicable; in vitro test

Control animals:

other: not applicable, in vitro test

Positive control reference chemical:

As positive control 1 μM verapamil was incubated in parallel to the test item (n=3), and the depletion of the compound was monitored to demonstrate the enzymatic activity of the S9 fractions. Positive control samples were taken after 0 and 30 minutes.

Details on study design:

- Dose selection rationale: based on experimantal method development (analytical detection)

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Time and frequency of sampling: after 0, 2, 5, 10, 15 and 30 minutes


METABOLITE CHARACTERISATION STUDIES
- Method type(s) for identification: Liquid chromatography – mass spectrometry (LC-MS)
- Limits of detection and quantification:
- Other: after incubation time samples were samples were processed for ACN precipitation and quantitative bioanalysis (addition of two volumes (i.e. 400 μl) stop solution (ACN containing the ISTD).

Statistics:

Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the database were conducted using Microsoft Excel.

Results and discussion

Main ADME results

Type:

metabolism

Results:

The ester was rapidly converted into MAA and the respective alcohol. (<0.25 min)

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Methacrylic acid (MAA) and benzyl alcohol

Any other information on results incl. tables

As shown in the Table 2, already at the first time point 0 min (+0.25 min), only 0.3 μM BNMA was detectable which corresponds to 1.5% of initial BNMA concentration. At the same time, the analytical measurements revealed the formation of the two metabolites Benzyl alcohol (Table 3) and Methacrylic acid (only qualitative detection of MAA). These findings clearly indicate that BNMA was rapidly metabolised within the first minute.

Table 2: Remaining BNMA (nominal initial concentration: 20 µM): measured concentration and calculated percentage of remaining test item after incubation with rat liver S9 fractions for different time points, (n=3)

Remaining BNMA concentration				% remaining BNMA of initial concentration
Time [min] (±0.25 min)	Mean (nM)[1]	SD (nM)	%CV[2]	Time [min] (±0.25 min)	Mean (%)
0	301.4	120.0	39.8	0	1.5
2	0.0	0.0	n.a.	2	0.0
5	0.0	0.0	n.a.	5	0.0
10	0.0	0.0	n.a.	10	0.0
15	0.0	0.0	n.a.	15	0.0
30	0.0	0.0	n.a.	30	0.0

 

Table 3: Measured concentration and calculated percentage of formed metabolite benzyl alcohol after incubation of BNMA (nominal initial concentration: 20 µM) with rat liver S9 fractions for different time points, (n=3)

Formed Benzyl alcohol concentration				% formed Benzyl alcohol of initial concentration in BNMA
Time [min] (±0.25 min)	Mean (nM)	SD (nM)	%CV	Time [min] (±0.25 min)	Mean (%)
0	22385.6	2407.1	10.8	0	111.93
2	21714.8	2761.8	12.7	2	108.57
5	22218.3	1915.1	8.6	5	111.09
10	19567.2	423.3	2.2	10	97.84
15	20073.7	3283.2	16.4	15	100.37
30	23577.3	5884.2	25.0	30	117.89

[1]values were at or below quantification limit (LOQ 400 nM)

[2]Coefficient of variation expressed as percentage of SD divided by mean value

Taken all findings together it can be concluded that 20 μM BNMA was completely metabolized within very short time (< 0.25 min; at time point 0 min) in presence of 0.5 mg/ml rat liver S9 fractions (i.e. in presence of phase I and II enzymes). At time point 0 min already no BNMA was detectable and the formation of the primary metabolites could be shown as a proof of the very rapid enzymatic hydrolysis of BNMA. Due to the immediate cleavage of BNMA in presence of rat liver S9 fractions, half-life and intrinsic clearance could not be calculated.

Applicant's summary and conclusion

Conclusions:

Taken all findings together it can be concluded that 20 μM BNMA was completely metabolized within very short time (< 0.25 min; at time point 0 min) in presence of 0.5 mg/ml rat liver S9 fractions (i.e. in presence of phase I and II enzymes). At time point 0 min already no BNMA was detectable and the formation of the primary metabolites could be shown as a proof of the very rapid enzymatic hydrolysis of BNMA. Due to the immediate cleavage of BNMA in presence of rat liver S9 fractions, half-life and intrinsic clearance could not be calculated.

Executive summary:

Taken all findings together it can be concluded that 20 μM BNMA was completely metabolized within very short time (< 0.25 min; at time point 0 min) in presence of 0.5 mg/ml rat liver S9 fractions (i.e. in presence of phase I and II enzymes). At time point 0 min already no BNMA was detectable and the formation of the primary metabolites could be shown as a proof of the very rapid enzymatic hydrolysis of BNMA. Due to the immediate cleavage of BNMA in presence of rat liver S9 fractions, half-life and intrinsic clearance could not be calculated.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.